Domestic dogs were reported to have about 213 cases of cancer annually per 100,000 individuals in a survey performed several decades ago, which was similar to the incidence reported in humans at that time (approximately 300 per 100,000) (1). Although more recent studies of cancer incidence in dogs are not available, it is likely that the current incidence is even higher, as dogs are living much longer than they did decades ago. The development of spontaneous tumors in dogs more closely resembles tumorigenesis in humans than does experimentally induced cancer in rodent models, and as such, there is great interest in using dogs with cancer for translational research. Several recent clinical trials have validated the use of spontaneous canine tumors for the preclinical evaluation of novel therapeutics (2-4). Unfortunately, there are relatively few noninvasive methods to diagnose cancer and monitor therapy in veterinary medicine when compared to human medicine. Development of novel assays for these parameters would be useful for both initial evaluation of canine cancer patients as well as for following these patients non-invasively during the course of a clinical trial.
Measuring circulating plasma or serum DNA as a biomarker in humans with cancer has gained attention in recent years, but has not been investigated in veterinary species. Circulating cell-free DNA is detectable at low levels in the serum and plasma of healthy humans, and concentrations of circulating DNA are increased in many human cancer patients. This increase is seen in multiple tumor types, including hematopoietic tumors (e.g. lymphoma and leukemia), carcinomas (e.g. lung, breast, cervical, pancreatic and gastrointestinal tumors), and other tumor types such as Ewing's sarcoma, melanoma, and glioma (5-9). It has been reported that about 50% of all human cancer patients have increased circulating DNA (10). Furthermore, many of the same genetic and epigenetic changes are present in circulating DNA and in DNA from primary tumors, including mutations and/or aberrant promoter hypermethylation in p53, p16, and APC genes (8,11-21). These findings indicate that at least a portion of circulating DNA is derived from tumor cells. In humans, a portion of circulating DNA is derived from the primary tumors (11,14,16-18,21,39). It is noted, however, that circulating DNA also is increased in many non-lymphoid neoplasms in humans (5-10,12,19,20,43).
Circulating DNA in cancer has been explored in humans as a screening tool and a prognostic indicator, as well as for detection of residual disease after treatment. Two approaches for these analyses are either absolute quantification of circulating DNA or evaluation of specific molecular genetic defects (i.e. tumor-specific mutations or epigenetic changes) in the circulating DNA. Increased plasma DNA and/or genetic and epigenetic changes in plasma DNA have been associated with poor prognosis in a variety of tumor types (6,17,18). For example, mean plasma DNA levels in patients with lung cancer (even stage 1a disease) are higher than healthy controls, and increased DNA in pre-treatment plasma samples correlated with decreased survival in patients with non-small cell lung cancer (5,22). Plasma DNA concentrations also are increased in patients with both early and late stages of lymphoma, and circulating tumor-derived DNA was detected by PCR in 86% of patients with B cell leukemia or lymphoma (12,23). Persistence of circulating tumor DNA after initiation of treatment was associated with poor treatment response or early relapse in these patients (23).
In another study K-ras mutations were present in the plasma of 17 of 21 pancreatic cancer patients. Importantly, the mutation in plasma DNA was identified 5 to 14 months before clinical diagnosis in 4 patients with no evidence of neoplasia on initial biopsy but who had pancreatic carcinoma confirmed on later histopathology (15). Lastly, changes in circulating DNA correlate with the presence of residual disease after initiation of treatment (24,25). It is important to note, however, that increases in circulating DNA are not specific to cancer, and have been reported in inflammatory and autoimmune diseases, as well as other conditions (26-29). Exogenous DNA, including viral DNA and fetal DNA, also can be detected in circulation (24,30-32).
The phenomenon of circulating tumor DNA has rarely been explored in laboratory animals. Subcutaneous injection of cultured tumor cells into rats allowed detection of tumor-specific plasma DNA 3 weeks after inoculation (33). To our knowledge there have been no studies evaluating circulating DNA in spontaneous tumors of veterinary cancer patients.